Photopharmacology of ion channels, adenosine receptors and myosin-V

(1)

Dissertation zur Erlangung des Doktorgrades

an der Fakultät für Chemie und Pharmazie

der Ludwig-Maximilians-Universität München

Photopharmacology

of Ion Channels, Adenosine

Receptors and Myosin-V

Katharina Hüll

aus

München, Deutschland

2020

(2)

(3)

Erklärung

Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Dirk Trauner betreut.

Eidesstattliche Versicherung

Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet.

München, 24.02.2020 Katharina Hüll

Dissertation eingereicht am: 16.09.2019

1. Gutachterin / 1. Gutachter: Prof. Dr. Dirk Trauner 2. Gutachterin / 2. Gutachter: Dr. Oliver Thorn-Seshold Mündliche Prüfung am: 5.11.2019

(4)

(5)

Acknowledgements

First and most importantly, I want to thank Prof. Dr. Dirk Trauner. I appreciated the tremendous amount of freedom and independence we are granted in your lab. Working for you has not only made me become a better scientist, but also the stronger person I am today. Our move to New York City came as a surprise but taking the risk has been the best decision of my life.

Second, I would like to thank Dr. Oliver Thorn-Seshold, who agreed to be the second advisor for this thesis.

I thank my committee: Prof. Dr. Konstantin Karaghiosoff, Prof. Dr. Oliver Trapp, Prof. Dr. Lena Daumann and Prof Dr. Thomas Klapötke

I thank the Studienstiftung des deutschen Volkes for their financial support and all the training and interaction with other students I was able to enjoy.

None of the work anyone of us does would be possible without the help of the permanent staff. Heike, Aleks, Danielle, Martin, Carrie, Mariia and Luis: thank you so much for making our lives easier.

Along these lines I would also like to thank the following people at LMU: Dr. Werner Spahl, Sonja Kosak, Dr. David Stephenson, Claudia Ober, Oliver Möbus-Ohly, Roland Schürer, Michael Gayer and Heidi Buchholz and at NYU: Dr. Chin Lin, Ron McLurkin and Philip “Pip” Morton.

My work would not have been possible without my mentors and close friends Laura and Matthias. Thank you for your loyalty and your support.

Reliable colleagues and collaborators are extremely important, and I would like to thank Martin Maier, Dr. Bryan Matsuura and Dr. Timm Fehrentz for being so great to work with.

Thank you to all the talented students I had the chance to work with: Lucie Thomas, Benjamin Heinz, Solène Beauchamp, Meike Amma, Mandira Banik and Chris Arp. Working in the lab is always most enjoyable with the right people around, and I have been lucky to experience both Munich (Green Lab and BioSysM) and NYU labs and I enjoyed and appreciated all of them. The list of people I shared lab space with is endless, but you guys were the reason I came to lab in the morning with a smile on my face.

In particular, thank you, Nina, Felix, Daniel, Philipp, Giulio, Arunas, Julius, Martin M. and Anna I.: You have been more than just colleagues, thank you for being my friends. And most importantly, thank you, Peter, Ally, Dylan, Tom, Max and Steve: you made New York City feel like home.

I would be nothing without my friends. Thank you Krissi, Adri, Krisi, Miri, Angie and Nina: From first year-chemistry students until today, we somehow have grown into

(6)

vacations and that you share your world, the real world, with me. Thank you Stuffi, Fränzi und May: My school years were much more bearable with you, and now we are in our 30s and life is still better when you are around.

Thank you, Anna. I am so happy we are finally neighbors again.

I am most grateful to my parents. Your unconditional support and love are the reason I am where I am today.

(7)

Project Affiliation Disclosure

Apart from myself, many collaborators, colleagues and students have worked hard for the results presented in this thesis. In this statement I proclaim that the following findings were a team-driven effort and that I am compiling and discussing our findings. Alongside the supervision of Prof. Dr. Dirk Trauner, the following people were involved. Results that have been published in or submitted to a peer-reviewed journal include an author list at the beginning of the subchapter as well.

Chapter I: Johannes Morstein (chapters on transporter/pumps, enzymes and other applications)

Chapter II: Dr. Julie Trads (Synthesis and Electrophysiology), Dr. Bryan Matsuura (Synthesis), Dr. Laura Laprell (Electrophysiology), Dr. Timm Fehrentz (Electrophysiology), Dr. Nicole Görldt (Electrophysiology), Dr. Krystian Kozek (Thallium Flux Assays), Prof. Dr. David Weaver (Supervision), Prof. Dr. Nikolaj Klöcker (Supervision), Dr. David Barber (Synthesis and Supervision), Martin Maier (Synthesis and Computational Analysis), Martin Reynders (Synthesis), Philipp Leippe (Synthesis), Tongil Ko (Synthesis), Lukas Schäffer (Synthesis)

Chapter III: Tyler Benster (Computational Analysis), Prof. Dr. Michael Manookin (Electrophysiology), Prof. Dr. Russell Van Gelder (Supervision), Dr. Laura Laprell (Computational Analysis, Electrophysiology and Supervision)

Chapter IV: Dr. Matthias Schönberger (Synthesis and Supervision), Dr. Daniela Malan (MEA with Cardiomyocytes), Prof. Dr. Philipp Sasse (Supervision), Prof. Dr. David Weaver (Thallium Flux assays and Supervision), Prof. Dr. Bryan Roth (PDSP Binding Studies),

Chapter V: Solène Beauchamp (Synthesis)

Chapter VI: Lucie Thomas (Synthesis), Benjamin Heinz (Synthesis), Georg Merck (Single Molecule Tracking), Prof. Dr. Zeynep Ökten (Supervision)

Chapter VIII: Meike Amma (Synthesis), Kevin Sokol (Synthesis), Christopher Arp (Synthesis)

I am very grateful to have met all these people and that I was able to work with and learn from them.

(8)

(9)

Publications

Parts of this doctoral thesis have been published in scientific journals, or are considered for publication, respectively:

1. Maier, M.; Hüll, K.†_{; Reynders M.}†_{; Matsuura, B.}†_{; Leippe, P.; Ko, T. Schäffer, L.}

and Trauner, D. An Oxidative Approach Enables Efficient Access to Cyclic Azobenzenes, J. Am. Chem. Soc. 2019, 141, 17295–17304.

2. Trads, J. †_{; Hüll, K.} †_{; Matsuura, B.} †_{; Laprell, L.; Fehrentz, T.; Görldt, N.; Kozek,}

K. A.; Weaver, D.; Klöcker, N.; Barber, D.; et al. Sign Inversion in Photopharmacology: Incorporation of Cyclic Azobenzenes in Photoswitchable Potassium Channel Blockers and Openers. Angew. Chem. Int. Ed. 2019, 58,

15421-15428.

3. Hüll, K.; Benster, T.; Manookin M.; Trauner, D., Van Gelder, R. N.; Laprell, L. Photopharmacologic Vision Restoration Reduces Pathological Rhythmic Field Potentials in Blind Mouse Retina, Sci. Rep. 2019, 9,13561.

4. Hüll, K.†_{; Morstein, J.}†_{; Trauner, D. In Vivo Photopharmacology. Chem. Rev.}

2018, 118, 10710–10747.

†_{: equal contribution}

Further publications in peer-reviewed journals, which are not part of this thesis are: 5. Laprell, L.; Hüll, K.; Stawski, P.; Schön, C.; Michalakis, S.; Biel, M.; Sumser, M. P.; Trauner, D. Restoring Light Sensitivity in Blind Retinae Using a Photochromic AMPA Receptor Agonist. ACS Chem. Neurosci. 2016, 7, 15–20.

6. Damijonaitis, A.; Broichhagen, J.; Urushima, T.; Hüll, K.; Nagpal, J.; Laprell, L.; Schonberger, M.; Woodmansee, D. H.; Rafiq, A.; Sumser, M. P.; et al. AzoCholine Enables Optical Control of Alpha 7 Nicotinic Acetylcholine Receptors in Neural Networks. ACS Chem. Neurosci. 2015, 6, 701–707.

(10)

(11)

Abstract

Over the past decade, photopharmacology has become an established method to address biological questions. From the first proof-of-principle that the small molecule azobenzene can be used to control the function of much larger biomolecule like transmembrane receptors, the field has rapidly developed and is now providing tools for medical research.

In this work, we have synthesized and characterized photoswitchable ligands for GPCRs, ion channels, and the cytoskeleton, including adenosine receptors (AzoAdenosines), ryanodine receptors (AzoCaffeines), voltage-gated ion channels (various open channel blockers, KCNQ channel modulators and arsenic analogs for co-crystallization) and myosin V (AzoMyoVIns).

In particular, we have focused on the development and characterization of new photoswitches and their biochemical or medical application. We disclosed an efficient access to cyclic azobenzenes (diazocines) and have thoroughly investigated the effects of different substitution patterns on their photophysical properties. These photoswitches are stable in their bent cis-form, and we could show that using these new types of photoswitches, enables inversion of the biological activity of the photoswitch, compared to an azobenzene analog (Figure 1).

Figure 1: The concept of sign inversion in photopharmacology using diazocine photoswitches.

(14)

This concept was demonstrated with openers of G protein-coupled inwardly rectifying potassium (GIRK) channels and blockers for voltage-gated potassium (KV)

channels. While azobenzene-based GIRK-channel openers and KV channel

blockers bound to their target in the dark-state (E-form), our diazocine-based analogs bound upon illumination with light (also E-form). This orthogonal toolset will enable researchers to tailor their photoswitch design according to the desired application.

Additionally, we have continued to develop new azobenzene-based photoswitchable ion channel blockers for vision restoration purposes. Our molecules conferred light sensitivity to degenerated and blind mouse retinas, as shown in multi-electrode array studies. Focusing on low-frequency oscillations in the retina (local field potentials, LFPs), which develop during the degeneration process, we discovered a unique property of azobenzene photoswitches. Upon switching, they suppress these detrimental oscillations and lead to an improved signal to noise ratio, a characteristic which makes them unique among other vision restoration approaches, like electrical implants or gene therapy.

(15)

(16)

(17)

(18)

(19)

In Vivo Photopharmacology

Katharina Hüll§_{, Johannes Morstein}§_{, and Dirk Trauner}*

Department of Chemistry, New York University, 100 Washington Square East, New York, NY 10003-6699, USA.

§_{These authors contributed equally}

Abstract

Synthetic photoswitches have been known for many years but their usefulness in biology, pha6,7_{rmacology, and medicine has only}

recently been systematically explored. Over the last decade photopharmacology has grown into a vibrant field. As the photophysical, pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biological targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), soluble proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, we provide an overview of photopharmacology using synthetic switches that have been applied in vivo, i.e. in living cells and organisms. We discuss the scope and limitations of this approach to study biological function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.

(20)

Contents – In Vivo Photopharmacology

ABSTRACT

1 INTRODUCTION

2 PHOTOSWITCHES FOR ION CHANNELS

2.1 Ionotropic Glutamate Receptors 2.1.1 AMPA receptors

2.1.2 Kainate Receptors 2.1.3 NMDA Receptors

2.2 Pentameric Ligand-Gated Ion Channels 2.2.1 Nicotinic Acetylcholine Receptors (nAChRs) 2.2.2 GABAA Receptors

2.3 Voltage-Gated Ion Channels 2.4 Other Ion Channels

2.4.1 TRP Channels

2.4.2 ATP-Sensitive Potassium Channels

2.4.3 G Protein-Coupled Inwardly Rectifying Potassium Channels 2.4.4 ENaC/P2X Superfamily

3 PHOTOSWITCHES FOR TRANSPORTERS AND PUMPS

4 PHOTOSWITCHES FOR GPCRS

4.1 Class A, Rhodopsin-Like

4.2 Class B, Secretin Receptor Family

4.3 Class C, Metabotropic Glutamate Receptor Family

5 PHOTOSWITCHES FOR ENZYMES

5.1 Kinases 5.2 Phosphatases 5.3 Proteases

5.4 Histone Deacetylases and Histone Methyltransferases 5.5 Acetylcholinesterases

5.6 RNA Polymerases

5.7 DNA Gyrases and Dihydrofolate Reductases 5.8 Lipoxygenases

(21)

5.9 Nitric Oxide Synthases 5.10 Receptor-Linked Enzymes

6 PHOTOSWITCHES THAT TARGET THE CYTOSKELETON

7 OTHER APPLICATIONS OF PHOTOSWITCHES

7.1 Membrane Transport 7.2 Protein Translation 7.3 Cytotoxicity

7.4 Immunobiology

7.5 Cell Adhesion and Cell Communication

8 PHOTOPHARMACOLOGY WITH SYNTHETIC SWITCHES VS.

ALTERNATIVE APPROACHES FOR OPTICAL CONTROL

9 CONCLUSION AND OUTLOOK

10 TABLE OF STRUCTURES

10.1 Photoswitches for Ion Channels

10.2 Photoswitches for Transporters/Pumps 10.3 Photoswitches for GPCRs

10.4 Photoswitches for Enzymes 10.5 Photoswitches for Cytoskeleton 10.6 Other Applications of Photoswitches

11 LIST OF ABBREVIATIONS

(22)

Introduction

Light is unsurpassed in its ability to control biological systems with high spatial and temporal resolution. It has the advantages of non-invasive and remote action, reversibility, speed, and facile modulation of the energies involved. This has been recognized in optogenetics, a field that has grown from an attempt to optically control neuronal activity to a broad effort to use light as a precision tool in biology.1

In its most broadly used form, optogenetics relies on naturally occurring chromophores, such as retinal or flavins, to convey the interaction of light with its receptors on a molecular and cellular level.

Light, however, can also influence the biological activity of synthetic molecules by changing their pharmacokinetic or pharmacodynamic properties. This combination of photochemistry and pharmacology is now known as “photopharmacology”. While other names, such as “optopharmacology” and “chemical optogenetics”, have been suggested, in our opinion, photopharmacology is the most suitable one, both for etymological and for esthetic reasons.

The effect of light on small molecules can be irreversible or reversible and both modalities have been used in biology.2_{The irreversible photochemical inactivation}

of drugs, for instance, has been occasionally applied to probe the functional role of their biological targets (Fig. 1A).3_{Photocleavable protecting groups (“cages”)}

have enabled the conditional release of biologically active ligands (Fig. 1B). To date, caged ligands are arguably the most widely used photopharmacological tools and new varieties of photocages continue to emerge.4,5_{However, caged}

ligands have been extensively reviewed elsewhere and are therefore not a subject of this article.5,6_{Other modes of light-activation (e.g. PACT) can be considered but}

are beyond the scope of this review.7

Here, we focus on synthetic photoswitches that allow for the fast and reversible optical control of biological systems and we will only cover switches that have been used in vivo. As far as this review is concerned, in vivo means in a living cell (‘in

cellulo’) and in an animal (ideally awake and with quantifiable behavior). In

comparison to molecular targets investigated in vitro, cellular systems exhibit physically permeable barriers, compartmentalization, and a crowded and complex target environment.8,9_{Within the last years, the use of photopharmacology in}

livings systems has grown considerably and its applicability in biology and complementarity to conventional optogenetics has become evident. The timeliness of in vivo photopharmacology is also evident by the fact that nearly half of the contributions reviewed here were published in 2015 or thereafter. We refer the reader to other comprehensive reviews on photopharmacology for an overview of in vitro studies.10,11

(23)

Synthetic photoswitches can be classified by the mechanism with which they interact with their targets (Fig. 1C-F). So-called Photochromic Ligands (PCLs) are freely diffusible molecules that can adapt two (or more) different isomeric forms upon irradiation with light, which exhibit different affinities and/or efficacies towards their biological targets and have different pharmacodynamics (Fig 1C). Switching can also affect the bioavailability (pharmacokinetics) of the PCL.

In a second modality, ligands can be covalently tethered to the receptor through some form of bioconjugation, either to a native or to an engineered residue.12_This

allows for genetic encoding but also accelerates the response due to the high local concentration and the inability of the ligand to diffuse away. In general, tethered photopharmacology is faster than photopharmacology with a freely diffusible ligand, which in turn is faster than normal pharmacology. Tethered ligands can function as agonists, inverse agonists, antagonists, or blockers pending on their pharmacology and/or their site of attachment.

Tethered ligands can be further divided into two subcategories depending on the closeness or remoteness of the covalent attachment site with respect to the ligand binding site: a) Photoswitchable Tethered Ligands (PTLs), and b) Photoswitchable Orthogonal Remotely Tethered Ligands (PORTLs).

If the photoswitch is mounted closely to the binding site and if it constitutes a large portion of the tether, as it is the case in a PTL, switching primarily effects the local concentration of the pharmacophore (Fig. 1 D). Ideally, the ligand cannot reach the binding site in one configuration and binds effectively in the other. Close tethering requires small bioconjugation motifs, e.g. a single cysteine point mutation. By contrast, the photoswitch in a PORTL mostly affects the efficacy of the tethered ligand and has a less pronounced effect on its local concentration (Fig. 1 E).13_Here,

the bioconjugation motif can be much larger, e.g. a self-labeling protein tag that reacts with the speed and selectivity of an enzymatic reaction (Halo, Snap, Clip tag etc.). These orthogonal tags can be fused with a protein of interest but can also be mounted onto an auxiliary protein within the same cell or even in an adjacent cell provided the tether is long enough and the local concentration can be kept high.14

Of course, the categories of close and remote tethering represent extremes and it is conceivable that there are tethered ligands which fall in between.

Another class of photoswitches covered in this review is light-responsive cross linkers (Fig. 1 F). Here, a photoswitch is covalently attached on both of its ends and, upon isomerization, influences the conformation and activity of a biological target (Fig 1 E). This requires two conjugation motifs on the biomolecule, e.g. two reactive cysteines appropriately spaced.

(24)

Figure 1: Modalities of photopharmacology. (A) Irreversible photoinactivation. (B)

Irreversible photoactivation (photo-uncaging). (C) Reversible photoactivation/ inactivation using a photochromic ligand (PCL) that toggles between an inactive (pentagon) or active (star) form. (D) A photoswitchable (closely) tethered ligand (PTL). (E) A photoswitchable orthogonal remotely tethered ligand (PORTL). (F) A photoswitchable cross-linker.

Other modalities are possible, such as photoswitches that are incorporated through expansion of the genetic code. Although this is strictly speaking not photopharmacology, the photoswitchable amino acid must be added externally and the photoreceptor so formed is functionally related to those covered in this review.

Having settled on a modality, the question arises as to which synthetic photoswitch to use. It must fulfill several requirements to be applicable in vivo: It should have favorable pharmacokinetics and should be metabolically stable in a given milieu. It cannot be phototoxic, which can occur when intersystem crossing competes with photoswitching.15_{Under physiological conditions, this can lead to the formation of}

singlet oxygen, which bleaches the chromophore and can be damaging to cells.16,17_{Moreover, the photoswitch should exhibit useful photophysical}

properties, such as high absorbance and quantum yields, and useful thermal relaxation rates. A B C D E F effect h ₁ h2or kBT effect h1 h2or kBT or kBT effect h₁ h2or kBT effect h₁ h2or kBT or kBT effect h effect

X

h

(25)

Figure 2: Synthetic photoswitches used in photopharmacology. The active

photoisomer is drawn on the left and the inactive photoisomer on the right. (A) An azobenzene that is thermally bistable and can be toggled between the active and inactive form using different wavelengths of light. (B) An azobenzene photoswitch that loses activity in the absence of light. (C) A bistable diarylethene photoswitch. (D) A hemithioindigo switch that is inactive in the dark and can be switched on with light.

Azobenzenes fulfill most of these criteria and therefore account for the majority of switches used to date, although diarylethanes, fulgides, and hemithioindigos have been occasionally employed (Fig. 2).18–22_{Therefore, some functional features of}

molecular photoswitches are best discussed with azobenzenes. Since the absorption spectra of their isomeric forms overlap to a certain degree one must consider photostationary states, which are a function of the wavelengths used. The isomeric ratio in the photostationary state can approximate 99:1 if the absorption spectra are very well separated but is usually much lower, e.g. 80:20. Due to the inherent non-linearity of biological systems, however, these low ratios can still be

(26)

highly consequential. Photoswitches that are ineffective in an in vitro enzymatic assay and show considerable background activity can indeed have pronounced and clean effects in a physiological experiment.

The photochemical interconversion of isomers is overlaid by thermal relaxation, i.e. the ability of the switch to fall back into its thermodynamically more stable form. The rate of this thermal relaxation depends on the electronic structure of the azobenzene and is also influenced by temperature and solvent. As far as in vivo photopharmacology is concerned, the solvent is an aqueous solution at physiological pH and with high ionic strength, which lowers the barrier for thermal isomerization.23_{Nevertheless, some azobenzene switches remain stable in their}

high-energy isomeric form for hours or days once the light is turned off,24–26

whereas others undergo very fast thermal relaxation in the dark.27,28_{Depending on}

the biological system investigated and its responsiveness, either bistability or the automatic deactivation of a fast relaxing photoswitch can be desirable.29_The

former would be the case, for instance, with photopharmacology that targets gene expression, whereas the latter is applicable in neural systems with millisecond response times, such as the human retina.

With these considerations in mind and a focus on biological applications, we have organized this review according to the biological targets amenable to in vivo photopharmacology. These include ion channels, transporters and pumps, GPCRs, enzymes, elements of the cytoskeleton, and a few examples, which go beyond these target classes. In the concluding section, we will compare photopharmacology with other methods for controlling biological activity with light and discuss the advantages and disadvantages of synthetic switches, caged compounds, and optogenetic approaches that involve natural chromophores. Lastly, we will briefly discuss the state of the art and future directions of photopharmacology with respect to clinical applications.

(27)

Photoswitches for Ion Channels

Ion channels are involved in fast synaptic transmission and play a key role in nervous systems. They are also important for secretory processes and the maintenance of body homeostasis. They have highly developed pharmacology and their fast kinetics are well matched to the kinetics of synthetic photoswitches. As such, photopharmacology has been particularly successful with ion channels.

Ionotropic Glutamate Receptors

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels, which open upon activation with presynaptically released glutamate and trigger action potentials in postsynaptic neurons. Based on their affinity towards certain agonists and their genomic sequence, iGluRs have been divided into AMPA (iGluAs), kainate (iGluKs) and NMDA (iGluNs) receptors. Due to their fundamental role in neural processing and extensive biophysical characterization, iGluRs emerged as one of the first targets for photopharmacology.

AMPA receptors

In 2012, Trauner and colleagues introduced the photoswitchable agonist ATA (Table 1, #1), which was characterized in mouse brain slice and HEK293T cells.30

ATA activated GluA2 receptors in its trans-form and could be inactivated with blue

light. Accordingly, trains of action potentials (APs) were generated in the dark, while AP firing stopped upon irradiation with 480 nm light. In a subsequent study,

ATA was explored in the context of vision restoration.31_{Multi-electrode-array (MEA)}

recordings and patch-clamp electrophysiology carried out with degenerated mouse retinae revealed that ATA primarily acts on retinal ganglion cells (RGCs) and amacrine cells. Computational ligand docking studies showed that the trans isomer binds tightly and allows for full closure of the clamshell-like ligand binding domain of the receptor, whereas the cis-isomer rapidly dissociates.32

More recently, a photoswitchable antagonist for AMPA receptors, termed

ShuBQX-3 was developed, complementing the photopharmacology of AMPA

receptors (Table 1, #2).33_{ShuBQX-3 could be used to control action potential firing}

in hippocampal CA1 neurons with 460 nm/600 nm light. Interestingly, the absorption and action spectrum of ShuBQX-3 undergoes an unusually large bathochromic shift upon binding to the receptor or interacting with free arginine in solution. This large difference between the UV-Vis spectra in the absence of the receptor and the action spectra has been rarely observed in photopharmacology.

(28)

Kainate Receptors

Optical control over GluK receptors was achieved with the PCL GluAzo (Table 1, #3).34,35_{Signaling through native GluK1 and GluK2 receptors could be controlled}

with 380 nm/500 nm light. Photoswitching of iGluR-currents was demonstrated in transfected HEK293T cells, dissociated neurons and Purkinje cells.36

Covalent attachment of the glutamate photoswitch L-MAG-1 (Table 1, #4) to a cysteine mutant of GluK2 gave rise to the light-gated glutamate receptor, LiGluR (Fig. 3).37–39_{LiGluR could be monitored with electrophysiology (Fig. 3B and C) but}

also with calcium imaging due to its Ca2+_{-permeability.}40_{PTLs with longer}

(L-MAG-2; Table 1, #4) and shorter linkers (L-MAG-0; Table 1, #4) were also developed,41

as well as versions with red-shifted action spectra (L-MAG-0460; Table 1 #5 and

toCl-MAG; Table 1, #6).42,43_{Furthermore, two-photon (2P) switching could be}

demonstrated with MAGs.44,45_{To this end, MAG was modified with a linker (MAG}

2P; Table1, #7) and equipped with a 2P antenna (MAGA; Table1, #8).45_{The biophysical}

properties of LiGluR and its variants have been investigated in detail. For instance, the PTL was found to function as a photoswitchable full agonist.39_{These studies}

also provided insights into the function of the native receptor and helped to clarify receptor gating kinetics and its desensitization mechanism.46_{Depending on the}

point of mutation, L-MAG-0 can activate the receptor in either its cis- or its trans-form. This “yin/yang” behavior allows for independent activation of LiGluR in different subsets of neurons.

When expressed in sensory neurons of zebrafish larvae, LiGluR enabled optical control over their escape response. Photostimulation of Kolmer-Agduhr neurons with LiGluR provided insights into their physiological role in zebrafish.47_The

calcium permeability of LiGluR also allowed for the optical control of a variety of calcium-dependent biological processes, such as exocytosis,48_{neurotransmitter}

release in chromaffin cells,49_{and glutamate release in astrocytes through increased}

Ca2+_levels.50

In the retina, LiGluR can be selectively expressed via transfection with adeno-associated virus (AAV) and labeled with MAGs.51_{About five weeks after injection,}

LiGluR was found exclusively in RGCs. In multielectrode array (MEA) recordings with 5 s light/dark intervals, sustained light responses were observed, which declined in magnitude to reach a plateau after a few cycles. This was sufficient to restore the pupillary light reflex and light-avoidance behavior. With the development of red-shifted MAG-derivatives, in vivo application became more feasible. As a red-shifted azobenzene, L-MAG-0460 does not require irradiation with UV light and quickly turns off in darkness. MEA recordings with LiGluR-transfected, degenerated retinae from rcd1 dog model showed light responses of RGCs. In rd1 mice, in addition to expression in RGCs (Fig. 3D and E), LiGluR was transfected in ON-bipolar (ON-BP) cells using AAV and an ON-BP specific promoter.52_{Targeting bipolar cells upstream of RGCs makes it possible to exploit}

(29)

Figure 3: Ionotropic glutamate receptors. (A) Schematic depiction of LiGluR.

Isomerization of the photoswitch brings the active ligand (red star) in close proximity to the binding site, opening the channel. (B) iGluR6(L439C) transiently expressed in hippocampal neurons with L-MAG covalently attached show trains of AP firing upon illumination with 380 nm light. Illumination with 500 nm silences AP firing. Reprinted with permission from reference (38). Copyright 2007 Elsevier Ltd. (C) Neurons lacking LiGluR do not respond to alternating light but do respond to current injections. Reprinted with permission from reference (38). Copyright 2007 Elsevier Ltd. (D)-(E) Multi-electrode-recordings of retinal ganglion cells from blind mouse retinae, overexpressing LiGluR without (D) or with (E) L-MAG-0460. Alternating

dark/445 nm sequences leads to firing of retinal ganglion cells in a blind mouse model. Reprinted with permission from reference (52).

A B C D E h₁ h2or kBT

(30)

retinal cell processing above the level of RGCs. The resulting signal should resemble the wild type retina signal more closely. Although the MAGs worked well in RGCs, their hydrolytic instability limited their use in less accessible cells.53_This

prompted the development of PORTLs (see section 3.3).

Gorostiza and colleagues targeted native kainate receptors in the retina via affinity labeling with photoswitchable glutamate derivatives called TCP-9 and TCP-10 (Table 1, #9 and #10.) These PTLs bear an N-hydroxy succinimide ester (NHS-ester) as an electrophile, which reacted with native lysines close to the ligand binding site.54_{They were used to restore light-dependent electrical activity in a blind mouse}

retina, as demonstrated with MEA recordings. This approach is related to the photoswitchable affinity label concept (PAL) proposed for potassium channels and demonstrated with the enzyme human carbonic anhydrase.55,56_{Despite the}

instability of NHS-esters, maleimides, and related electrophiles, it holds promise for imparting light-sensitivity to native receptors with increased selectivity.

In addition to new MAG-derivatives, the incorporation of new GluK mutants expanded the repertoire of artificial photoreceptors.57_{The use of an optrode}

enabled the optical control of such LiGluR variants in awake, adult mice. The modularity of the glutamate receptor family was also exploited to engineer a light-gated K+_{-channel. Combining the K}+_{-selective pore domain of the bacterial}

glutamate receptor sGluR0 with the LBD of the GluK2 cysteine mutant gave rise to HyLighter.58_{HyLighter was expressed in cultured hippocampal neurons and}

conjugated with L-MAG-0 (Table 1, #4) to inhibit AP firing upon illumination. Expression in motorneurons of zebrafish larvae allows reversible suppression of escape behavior. Like its predecessor SPARK59,60_{(see below), HyLighter}58_provides

a solution to optical inhibition with a light gated potassium-selective channel, which has been difficult to achieve via engineering of channelrhodopsins.61

NMDA Receptors

With the development of the photoswitchable agonist ATG (Table 1, #11), photocontrol over the remaining glutamate receptor class – the NMDA receptors – was achieved in 2015.62_{ATG is bistable, active in its cis-form, and can be switched}

on with either UV light (370 nm) or with 740 nm 2P pulses. More recently, a photoswitchable antagonist, termed PNRA (Table 1, #12), for GluN receptors, was introduced.63_{This trans-active antagonist can be isomerized with 360 nm/420 nm}

of light and is the first antagonist displaying a higher affinity towards GluN2A and GluN2C over GluN2B and GluN2D.

Adopting the logic of LiGluR (i.e. LiGluKR), Isacoff and Trauner established that the photoswitches L-MAG-0 and L-MAG-1 (Table 1, #13) can be applied to NMDA receptors, giving rise to LiGluNR.64_{To this end, a small library of LiGluNRs with}

different cysteine mutants was generated and conjugated to the MAGs. Pending on the site of mutation and the PTL used, photo-agonism or photo-antagonism of

(31)

GluN2A and GluN2B could be achieved, enabling control of synaptic activation or spine-specific control over calcium levels. Transgenic zebrafish larvae were chronically NMDA-antagonized with short light flashes, leading to increased growth of retinal ganglion cells in their optic tectum.

Paoletti and colleagues incorporated the unnatural photoswitchable amino acid

PSAA (Table 1, #14) into GluN1, GluN2A, or GluN2B subunits to create light

sensitive NMDARs.65_{This work represented the first in vivo application of a}

photoswitchable amino acid integrated via genetic code expansion. Both activation and deactivation could be achieved upon UV-illumination, depending on the site of incorporation. Functional studies showed that sensitivity towards glutamate was not affected but the binding affinity of the co-agonist glycine could be modulated with light, thereby raising the open probability of the channel.

Pentameric Ligand-Gated Ion Channels

Nicotinic Acetylcholine Receptors (nAChRs)

Nicotinic acetylcholine receptors nAChRs are pentameric ligand-gated ion channels that are primarily permeable to sodium, potassium, and, in some cases, calcium ions. In 1969, the Erlanger group pioneered the photopharmacology of nAChRs, and the entire field of photopharmacology, by perfusing Electrophorous electroplaques with the photochromic ligands AzoCharCh (Table1, #15) and

azo-PTA (Table 1, #16).66_{Two years later, they published the PCL BisQ (Table 1, #17)}

and the covalently attached photochromic ligand QBr (Table 1, #18).67–70

Trans-BisQ proved to be a potent activator of Electrophorous electroplaques and

isomerized to the less active cis-isomer upon irradiation with 330 nm light. Erlanger, Lester and colleagues subsequently controlled the membrane potential of the squid giant axon with the azobenzene ammonium ion EW-1 (Table 1, #19)71,72_and

introduced the symmetrical photoswitchable antagonist 2BQ (Table1, #20).73

Many years later, Trauner and colleagues introduced a new PTL, AzoCholine (Table 1, #21). This compound, which was derived from phenyl choline ether, responded to 360 nm/440 nm light and was found to be a trans-active agonist for neuronal a7 nAChRs.74_{It provided optical control of the thrashing activity of} C. elegans nematodes. In the same study, BisQ was reevaluated and shown to

primarily act on muscular nAChRs, but not on neuronal nAChRs. Around the same time, Li and colleagues modified the insecticide imidacloprid, an agonist to insect nAChRs, with an azobenzene.75_{The resulting PCLs were tested for their insecticidal}

activity against the house fly Musca domestica. Under UV light irradiation, a decrease in LD50 by 80% was observed for the most active compound, AMI-10

(32)

Applying the PTL concept to nicotinic receptors, Trauner, Kramer, and colleagues designed the light-gated nicotinic acetylcholine receptor LinAChR using based on then available X-ray structure.76_{The PTLs MAACh (Table 1, #23) functioned as a}

photoswitchable agonist, whereas MAHoCh (Table 1, #24), proved to be a light-activated antagonist. Both PTLs respond to 380 nm/500 nm light and are bistable under physiological conditions. LinAChR was evaluated in Xenopus oocytes but is expected to work well in excitable cells and complex neuronal systems.

GABA

A

Receptors

GABAA receptors are chloride-selective pentameric ligand gated ion channels that

decrease the likelihood of action potential firing upon opening. In 2012, Trauner and colleagues published a light-switchable derivative of propofol, termed AP-2 (Table1, #25).77_{This compound functioned as an allosteric modulator that}

potentiated GABA currents in the dark and could be inactivated upon irradiation. The effect on chloride currents was investigated with Xenopus ooyctes overexpressing the a1b2g2-GABAA variant. Light dependent anesthesia was

demonstrated with Xenopus laevis tadpoles. Concurrently, Pepperberg and co-workers published a different series of photochromic propofol analogues, including the PCL MPC-088 (Table 1, #26) and the PTL MPC-100 (Table 1, #27).78

They applied these compounds to oocytes and to cerebellar slices and could modulate membrane current and spike firing rate in Purkinje cells using UV/blue light. Shortly thereafter, the Kramer group introduced another light-regulated GABA receptor (LiGABAR) using tethered photopharmacology.79_{In this case, they}

engineered a GABAA receptor bearing a cysteine mutation in its a1-subunit that

served as conjugation site for the PTL MAM-6 (Table 1, #28). Although it was derived from muscimol, MAM-6 does not act as a photoswitchable agonist but as an antagonist. The response of LiGABAR to its endogenous ligand can thus be modulated with light. Illumination at 380 nm allows for greater activation with GABA than at 500 nm, as the ligand antagonist retracted from the binding site in the cis-state of the switch. LiGABAR was used in Xenopus oocytes, HEK293T cells, and cultured hippocampal neurons, as well as in brain slices. The generation of a LiGABAR knockin mouse line allowed photocontrol of LiGABAR at endogenous expression levels.80_{In the course of this study, new PTLs with higher efficacy were}

introduced, e.g. the guanidinium analog PAG-1C (Table 1, #29). This refined version of LiGABAR enabled the optical control of cortical neurons in awake mice.

Voltage-Gated Ion Channels

Voltage-gated ion channels, which include potassium (KV), sodium (NaV), and

calcium (CaV) channels, play crucial roles in the generation of action potentials and

in synaptic transmission and are prime targets for photopharmacology. Not surprisingly, they have played a prominent role in the development of the field. In

(33)

2004, Trauner, Kramer, and Isacoff introduced a light and voltage sensitive potassium channel called SPARK.59_{To this end, they endowed a Shaker K}

V with an

engineered cysteine, which served as a bioconjugation motif for a tethered quaternary ammonium ion (MAQ, Table 1, #30). Isomerization of the photoswitch with 380 nm/500 nm light shortened and lengthened the tether, unblocking and blocking the pore. This translated to the reversible optical control of action potential firing in hippocampal pyramidal neurons. An additional mutation in the channel pore converted the potassium-selective SPARK into a non-selective ion channel (D-SPARK).60_{While SPARK channels led to the optical of neuronal}

inhibition, the unselective variant enabled light-controlled depolarization and excitation of dissociated neurons.

Photocontrol over the mechanically gated potassium channel TREK1 was achieved by conjugating MAQ (Table 1, #31) with a TREK1 cysteine mutant.81_{Isomerization}

with 380 nm/500 nm light resulted to reversible activation in HEK293T cells. Transfecting hippocampal neurons with the TREK1 mutant and labelling with MAQ allowed insights in the formerly unknown role of TREK1 in GABAergic signaling. While no light-elicited TREK1 currents could be detected at resting potential, baclofen-induced GABA currents could be modulated with light.

In addition to PCLs, cationic blockers that function as PTLs have been systematically explored (Fig. 4). Although these compounds are much less selective than MAQ as they affect a range of voltage gated ion channels, they have nevertheless proven to be powerful photopharmacological tools in vivo and they are on the verge of becoming clinically relevant. The first compound that functioned in this way was

AAQ (Table1, #32).55_{Although it was initially designed to attach itself covalently via}

affinity labelling, it proved to be a PCL that binds to the inner cavity of voltage gated ion channels and has long-lasting effects to cells due to its lipophilicity.82_{As such,}

it is a photoswitchable version of the well-known use-dependent open-channel blocker lidocaine. AAQ can be isomerized with 360 nm/500 nm light and is thermally relatively bistable. Suppression of AP firing could be achieved with 380 nm light, whereas 500 nm light restored neuronal activity. In a subsequent study, AAQ was used to convey light-sensitivity to blind retinae, where it mainly targets amacrine cells.83_{In addition to AAQ, a series of derivatives with different}

photochemical, thermal, and pharmacological profiles was synthesized and characterized in cells. Amongst these, the highly lipophilic switch PrAQ (Table1, #33) turned out to be cis-active, inverting the logic of the otherwise trans-active blockers.

The photoswitches discussed above required UV irradiation for trans/cis isomerization and were thermally bistable, which limited their utility for applications in vision restoration. To address these issues, a second-generation of red-shifted photochromic ion channel blockers was developed that had absorption maxima in the 420–460 nm range.84_{The lead compounds that emerged from this study,}

(34)

potassium channels expressed in HEK293T cells in a light-dependent fashion and could be turned on and off with visible light and fast thermal relaxation, respectively (Fig. 4B and C). They worked in hippocampal brain slices and were subsequently used in vision restoration.85–87_{DENAQ provided sensitivity to white light at}

relatively low intensities and restored light-guided behavior in blind mice in an open field assay. Like DENAQ, BENAQ acted primarily on RGCs and showed long-lasting effects due to its high lipophilicity.

Figure 4: Photochromic ion channel blockers. (A) Schematic depiction of a

photoswitchable, use-dependent open channel blocker (red star). Upon isomerization to its inactive form (pentagon), the blocker unbinds from the pore. (B) Light-dependent potassium current elicited by isomerization of DENAQ. Reprinted with permission from reference (84). Copyright 2011 American Chemical Society. (C) optical control of action potential firing in hippocampal neurons with PhENAQ. Reprinted with permission from reference (84). Copyright 2011 American Chemical Society. (D)-(E) Multi electrode recordings in mouse retinae. Treatment with DAD results in light-dependent network activity (blue = 460 nm, black = dark). Figure created by the authors from data in reference (94).

A symmetrically substituted azobenzene with a quaternary ammonium ion on each side, termed QAQ (Table 1, #36) was introduced shortly after its unsymmetrical predecessors.88_{QAQ responds to 380 nm/500 nm light, is stable in the dark and}

can block KV, NaV and CaV channels. This bivalent ion channel blocker is not

membrane-permeable and needs to be actively transported into the cell via TRPV1 channels or P2X receptors. It was therefore possible to selectively target TRPV1 expressing cells for the optical control of nociception. More bistable and slightly red-shifted ortho-substituted derivatives of QAQ were reported shortly thereafter (Table 1, #37 and #38).89_{The even more red-shifted blocker QENAQ (Table 1, #39)}

was developed to combine the pharmacokinetics of QAQ with the photophysical

A B C D E + + + + + + + + + + + + + + +++ + + + + + + + h₁ h2or kBT

(35)

properties of DENAQ.90_{Like QAQ, QENAQ blocks Na}

V and KV and can be used to

optically control sensory DRG neurons.

The mechanism of action of DENAQ and BENAQ in degenerating retinae was unraveled in 2016 when Kramer and co-workers could show that they are subject to a similar transport mechanism as QAQ.86_{Like TRPV1, P2X receptors are known}

to dilate upon continued activation with ATP. As such, they can serve as an entrance gate in degenerating retinae. The authors suggested that this mechanism ensures that the photoswitch cannot be taken up into cells of a healthy retina, reducing side-effects. In vivo administration of these compounds requires intraocular injection, which is a standard procedure in ophthalmology, but carries a residual infection risk. Therefore, the slow release of QAQ and DENAQ from polymeric matrices was investigated.91

The permanently charged photoswitchable trimethylammonium ion AzoTAB (Table1, #40) had been used as a photoswitch in material science before it was developed into as photochromic ion channel blocker.92_{AzoTAB is isomerized from} trans to cis with UV light and can be switched back with blue light. In

cardiomyocytes, the trans isomer suppressed spontaneous electric excitability. The shape of action potentials indicated a trans block of fast NaV and CaV channels. In

more recent work, AzoTAB was shown to potentiate KV currents in the dark.93

Further studies culminated in the development of third-generation open-channel blockers, such as DAD (Table 1, #41).94_{In contrast to all previously described}

photoswitches, DAD does not bear a permanent charge and can rapidly diffuse across biological barriers in tissues. DAD can be isomerized with blue or white light and relaxes quickly in the dark. It mainly acts on bipolar cells exploiting the extant circuitry of a blind retina (Fig. 4D and E).

The photochromic blocker fotocaine (Table 1, #42),95_{another compound that is}

not permanently charged, was developed through “azologization” of the analgesic fomocaine. Azologization refers to the systematic replacement of isosteric motifs in drugs with azobenzenes.96_{Fotocaine can be isomerized with 350 nm/450 nm light,}

allowing and suppressing action potential firing in dissociated mouse hippocampal neurons.

In 2017, Berger and co-workers demonstrated that open-channel blocking of voltage-gated ion channels can also be achieved with guanidinium ions.97_They

synthesized an azobenzene version of the known open-channel blocker 2GBI,

photoGBI (Table 1, #43) to inhibit the voltage-gated proton channel HV1. The

trans-active blocker can be removed from the pore by illumination with blue light to induce proton currents in macrophages and sperm.

(36)

Other Ion Channels

TRP Channels

Transient receptor potential (TRP) channels are non-selective cation channels that are involved in the perception of pain, temperature, and pressure. They are multimodal and respond to a variety of input signals with the notable exception of light. The vanilloid receptor 1 (TRPV1) is one of the best studied members of this family (Fig 5). In 2013, Trauner and co-workers published the photoswitchable antagonists AC-4 (Table 1, #44) and ABCTC (Table1, #45), which responded to 360 nm/440 nm and 370 nm/470 nm light respectively.98_{They were evaluated in}

whole-cell patch-clamp experiments with HEK293T cells transiently transfected with TRPV1. Although both AC-4 and ABCTC inhibited the channel after voltage activation, only AC-4 antagonized the agonist capsaicin in a light-dependent manner. Moreover, AC-4 acts as a trans-antagonist on the voltage-activated channel and as a cis-antagonist in experiments with the agonist capsaicin.

The agonist capsaicin itself could be converted into a photoswitch by incorporating photoswitchable fatty acids (Fig. 5A). This yielded the azo-capsaicins (AzCAs) amongst which AzCA-4 (Table1, #46) turned out to be the most effective compound.99_{AzCA-4 operates with 350 nm/450 nm light and was used to optically}

control heterologously expressed TRPV1 in HEK293T cells and endogenous TRPV1 channels in dorsal root ganglions (DRG) cells, as assayed by electrophysiology and calcium imaging (Fig. 5B and C. Paving the way to photoanalgesics, AzCa-4 affected nociception of mouse C-fibers (Fig. 5D).

Peterson and colleagues achieved the optical control of a second member of the TRP channel family, TRPA1, with a small molecule termed Optovin (Table1, #47).100

This light-responsive rhodanine derivative activated the channel upon irradiation with violet light. The channel deactivated within seconds once the light was gone.

Optovin bestowed light sensitivity to endogenous TRPA1 channels in zebrafish

larvae, stimulating the dorsal fin of a spinalized fish. A subsequent paper presented various analogues that were used to control TRPA1b channels in zebrafish.101_They

provided light-control not only over sensory neurons, but also over the pace rate of zebrafish larvae hearts and human stem cell-derived cardiomyocytes.

More recently, Zufall and co-workers used photoswitchable diacylglyerols (PhoDAGs; Table1, #48) in the optical control of TRPC2 and TRPC6. Photosensitivity of TRPC2 and TRPC6 was demonstrated in cells and tissue slices in combination with Ca2+_-mapping.102_{For the optical control of TRPC3, Groschner}

and colleagues developed a new PhoDAG variant, termed OptoDArG (Table1, #49), which features two azobenzene containing acyl-chains.103_{It allowed for more}

efficient photoswitching of TRPC3 than PhoDAG-1 and provided insights into the lipid gating of this channel.

(37)

Figure 5: Photocontrol of TRPV1 channels. (A) Schematic depiction of TRPV1,

gated by a photochromic agonist (red pentagon/star). Opening occurs upon isomerization to the active isomer (red star). (B)-(C) light-dependent activation of TRPV1-channels expressed in HEK293T cells and treated with AzCa-4. Alternating UV and blue light in whole-cell voltage clamp (B) and current clamp (C) results in opening of the channel. The concentration of active photoisomer can be controlled by the wavelength (“color-dosing”). (D) Photocontrol of heat-sensitive C-fiber nociceptors with AzCA-4 in wild-type and knockout mice. Reprinted with permission from reference (99). Copyright 2015 Nature Publishing Group (https://doi.org/10.1038/ncomms8118, https://creativecommons.org/licenses/by/ 4.0/).

ATP-Sensitive Potassium Channels

ATP-sensitive Potassium Channels (KATP) consist of two distinct proteins, the

sulfonylurea receptor (SUR) and the inwardly rectifying potassium channel 6 (Kir6). Sulfonylureas, such as tolbutamide or glimepiride, mimic ATP and close KATP

channels, thus inducing insulin secretion from pancreatic b-cells. The photoswitchable sulfonylurea JB-253 (Table 1, #50), an azobenzene designed to structurally resemble glimepiride, closed KATP channels upon irradiation with blue

light, which was reversed upon thermal relaxation (Fig. 6A).19_{The effect of JB-253}

was only demonstrated in transiently transfected HEK293T cells (Fig. 6B), but also through the optical control of glucose stimulated insulin secretion in pancreatic islets (Fig. 6C and D). Isomerization to the active cis-isomer results in an increase of cytosolic Ca2+_{levels and insulin secretion. Subsequently, a red-shifted version of}

JB-253, JB-558 (Table 1, #51) was introduced.104_{This heterocyclic azobenzene}

absorbed maximally at 526 nm, could be isomerized with green light, and relaxed

C B A D h1 h2or kBT

(38)

Figure 6: Photopharmacology of KATP channels. (A) Whole-cell patch-clamp

recordings show that most efficient blocking of KATP with JB-253 occurred within a range from 440 nm to 500 nm light. Reprinted with permission from reference (19). Copyright 2014 Nature Publishing Group (https://doi.org/10.1038/ncomms6116, https://creativecommons.org/licenses/by/4.0/) (B)-(C) Optical control of human islets using JB-253. (B) Illumination of JB-253-treated b-cells resulted in rapid, reversible rises of cytosolic calcium levels in human tissue (Tb, tolbutamide; positive control). (C) Over 60% of cells respond to JB-253. (D) Representative cell-cell entrainment with identified hub (red). Reprinted with permission from reference numbers (19). Copyright 2014 Nature Publishing Group (E) Activation of a hub with

JB-253 and 470 nm light shows a larger number of entrained cells than activation of

rapidly in the dark. Binding to the SUR was confirmed by [3_{H]-glibenclamide}

displacement, and Epac2A signaling was optically controlled in FRET assays with an Epac2-camps biosensor. Like JB-253, JB-558 was able to induce glucose stimulated insulin secretion in its cis-form. In a combinatorial approach using

B A C D E x

(39)

photopharmacology and optogenetics, JB-253 was used to identify a subpopulation of cells in b-cell hubs that is required for regular insulin secretion (Fig. 6E and F).105_{Compounds like JB-253 could become valuable tools for the}

investigation of glucose stimulated insulin release and type 2 diabetes in vivo.106

Toxicological studies in various strains of S. typhimurium and E. coli with JB-253 indicated potential mutagenicity, but when administered orally to rats (1000 mg/kg over 7 days), no mortality and no apparent signs of toxicity were observed. Additionally, JB-253 exhibited metabolic stability, as demonstrated in an azoreductase assay. Light-induced remote activation of glucose homeostasis was demonstrated in mice using 50 mg/kg JB-253 and a 470 nm fiber optic cable placed in close proximity to the pancreas.

In insects, such as the cockroach Blattella germanica or the armyworm Mythimna

seperata, the SUR plays a crucial role in the inhibition of chitin biosynthesis. Optical

control of the SUR in this context was accomplished with a series of benzoylphenylurea (BPU) derivatives (Table 1, #52).107_{Insecticidal activity could be}

modulated with 365 nm and mortality rates increased upon irradiation.

G Protein-Coupled Inwardly Rectifying Potassium

Channels

G Protein-coupled inwardly rectifying potassium (GIRK) channels are an integral part of inhibitory signal transduction pathways. Opening of GIRK channels mediated by the Gbg subunit results in hyperpolarization of the cell membrane,

which reduces the activity of excitable cells. The extension of the selective GIRK channel opener ML-297 with an azobenzene led to a small library of light-operated GIRK channel openers (LOGOs).108_{These photochromic ligands could be}

isomerized using 360 nm/440 nm light. The compounds were validated in patch-clamp experiments with HEK293T cells overexpressing GIRK1/2 channels. The lead compound, LOGO-5 (Table 1, #53), was active in the dark and under 440 nm light and could be inactivated with 360 nm light. It was used to convey light-sensitivity to native GIRK channels and shape behavior in zebrafish larvae. Subsequently, a

LOGO derivative that fully responded to visible light, termed VLOGO (Table 1,

#54), was introduced.109_{This tetra-ortho-fluoro azobenzene was bistable and}

alternating illumination with 500 nm/400 nm afforded the optical control of hippocampal neurons.

ENaC/P2X Superfamily

Epithelial sodium channels (ENaCs) are heterotrimeric transmembrane proteins that control water transport across epithelia and are targeted by diuretics, such as amiloride. Extension of amiloride with an azobenzene yielded Photoamiloride-1 (PA-1; Table 1, #55), which was selective for the dbg subtype of ENaC.110_PA-1

(40)

functioned as a photoswitchable blocker in the trans-form responding to visible wavelengths of light (400 nm/500 nm). It was characterized using electro-physiology in Xenopus oocytes, HEK293T cells, and H441 cell monolayers. Due to its selectivity for dbgENaC, it could be used to distinguish between biological effects mediated by dbgENaC and abgENaC.

P2X receptors are cation-permeable, ATP-gated ion channels related to ENaCs. A strategy for their photoactivation exploiting the PTL concept was published by Grutter and co-workers in 2013.111_{Instead of ATP-dependent opening, the channel}

was endowed with light sensitivity through attachment of the photoswitches

MEA-TMA and MEA-TEA (Table 1, #56 and #57). The receptor could be turned on and

off with 365 nm/525 nm light, relying on a retractable ammonium ion to block the pore of the ion channel. Depending on the point of mutation, the channel pore could be blocked in the trans- or cis-state. Expression in dissociated hippocampal neurons allowed for the optical control of AP firing. North and colleagues crosslinked two subunits of the P2X2 receptor mutant with the azobenzene BMA (Table 1, #58) which allowed for rapid and reversible gating of the receptor with UV/blue light.112_{This modular approach was also applied to acid-sensing ion}

channels (ASICs), which are trimeric channels structurally similar to P2X. To further investigate the pore-opening of P2X channels, Grutter designed the longer photoswitchable cross-linker MAM (Table 1, #59), which could be switched with 365 nm/525 nm light.113

Photoswitches for Transporters and Pumps

Like ion channels, transporters and pumps facilitate the translocation of cargo across cell membranes. In contrast to the passive permeation through ion channels, however, this requires energy, which is either provided via ATP hydrolysis (primary active transport) of through the discharge of previously established gradients (secondary active transport). Photopharmacological studies have focused on neurotransmitter transporters providing an additional level of control over neural function. In 2014, Wanner and colleagues reported photoswitchable inhibitors of GABA transporters (Fig. 7A).114_{GABA-uptake assays in HEK293T cells showed that}

most of these are selective for GAT1. The lead-compound of the series (6e; Table 1, #60) enabled optical control of GABAA receptor-mediated currents in dentate

gyrus granule cells (Fig. 7B and C). While the cis-isomer prevented current flow, the

(41)

Figure 7: Optical control of a transporter. (A) Schematic depiction of the optical

control of a secondary active transporter with a photoswitchable inhibitor. (B) Light-induced activation of GAT1-mediated GABA uptake. Reprinted with permission from reference (114). Copyright 2014 American Chemical Society (C) Optical control of the excitatory amino acid transporter EAAT2. Reprinted with permission from reference (115). Copyright 2017 American Chemical Society.

In more recent work, a photoswitchable inhibitor of the glutamate transporter EAAT2 (excitatory amino acid transporter 2) was reported.115_{The design of this}

compound was inspired by the potent EAAT inhibitor of TFB-TBOA. A photoswitchable isoster, termed ATT (Table 1, #61), exhibited good selectivity for EAAT2 over other subtypes, as demonstrated with voltage clamp recordings in

Xenopus oocytes. The cis-isomer proved to be significantly less active as a blocker

than the isomer. Accordingly, irradiation of EAAT2 preincubated with

trans-ATT with 350 nm recovered transport currents.

Photoswitches for GPCRs

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome and are critically involved in signal transduction. They are

coupled to heterotrimeric G proteins, which, along with b-arrestin, mediate intracellular signaling. GPCRs have proven to be particularly amenable to photopharmacology, which may not be surprising since the only photoreceptors in humans, the opsins, are class A GPCRs that are covalently bound to a photoswitch,

viz retinal. A B dark 375 nm C h₁ h2or kBT

(42)

Class A, Rhodopsin-Like

In the 1980s, Erlanger, Wassermann and Lester showed that BisQ (Table1,#62), which was initially applied to nAChRs, also interacts with muscarinic acetylcholine receptors (mAChr) in frog hearts.116_{In addition to the PCL BisQ, its congener QBr}

(Table1, #63) was investigated as a tethered ligand.117,118_{Three decades later, the}

mAChRs were revisited by Decker and colleagues.119_{They developed BQCAAI}

(Table 1, #64), a bitopic (or dualsteric) photoswitchable ligand for M1 receptors based on iperoxo, a highly potent M1 agonist, and BQCA, a positive allosteric modulator. BQCAAI functions as a cis-antagonist and trans-agonist under illumination with 365 nm/455 nm light, respectively, when applied to HEK293T cells that express the receptor.

The optical control of µ-opioid receptors was achieved with a photoswitchable derivative of the highly potent agonist fentanyl (Fig. 8A).120_{Photofentanyl-2 (PF-2;}

Table 1, #65) functions as a bistable switch that is active in the dark, can be deactivated with 360 nm light, and reactivated with 480 nm light. The modulation of downstream signaling of the µ-opioid receptor via GIRK channels was demonstrated using patch-clamp electrophysiology in transiently transfected HEK293T cells (Fig. 8B and C).

Figure 8: Optical control of GPCRs. (A) Schematic depiction of the optical control

of the µ-opioid receptor (a Class A GPCR). Upon isomerization, the ligand (pentagon/star) activates the receptor and triggers a signaling cascade. (B) PF-2 is isomerized from cis to trans upon illumination with 480 nm, activating the µ-opioid receptor and inducing a GIRK-current via Gbg-signaling. Termination of the signal can